Research Article Consistent Rearrangement of Chromosomal Band 6p21 with Generation of Fusion Genes JAZF1/PHF1 and EPC1/PHF1 in Endometrial Stromal Sarcoma Francesca Micci,1 Ioannis Panagopoulos,4 Bodil Bjerkehagen,2 and Sverre Heim1,3 Departments of 1Cancer Genetics and 2Pathology, The Norwegian Radium Hospital; 3Faculty of Medicine, University of Oslo, Oslo, Norway; and 4Department of Clinical Genetics, University Hospital, Lund, Sweden Abstract Little is known about the genetic background of ESS as only 32 Endometrial stromal sarcomas (ESS) represent <10% of all such tumors have been karyotyped and reported scientifically uterine sarcomas. Cytogenetic data on this tumor type are (6–8). The pattern of rearrangements thus detected is nevertheless limited to 32 cases, and the karyotypes are often complex, clearly nonrandom with particularly frequent involvement of but the pattern of rearrangement is nevertheless clearly chromosome arms 6p and 7p (7). Recently, a specific translocation nonrandom with particularly frequent involvement of chro- t(7;17)(p15;q21) leading to the fusion of two zinc finger genes, mosome arms 6p and 7p. Recently, a specific translocation juxtaposed with another zinc finger (JAZF1) and joined to JAZF1 t(7;17)(p15;q21) leading to the fusion of two zinc finger genes, (JJAZ1), was described in a subset of ESS (9). Both genes, the JAZF1 at 7p15 and JJAZ1 at 17q21, contain sequences encoding zinc finger juxtaposed with another zinc finger (JAZF1) and joined to JAZF1 (JJAZ1), was described in a subset of ESS. We present motifs characteristic of DNA-binding proteins. The gene fusion three ESS whose karyotypes were without the disease-specific results in expression of a tumor-specific mRNA transcript V V t(7;17) but instead showed rearrangement of chromosomal containing 5 -JAZF1 and 3 -JJAZ1 sequences but retaining the zinc band 6p21, twice as an unbalanced t(6p;7p) and once as a finger motifs from both genes. Because wild-type JAZF1 is three-way 6;10;10 translocation. All three tumors showed expressed in normal endometrium, it has been suggested that the specific rearrangement of the PHD finger protein 1 (PHF1) JAZF1/JJAZ1 fusion gene creates a chimeric protein that disrupts gene, located in chromosomal band 6p21. In the two tumors transcription in a lineage-specific manner (9). with t(6;7), PHF1 was recombined with the JAZF1 gene from Several sarcoma-specific translocations exist, but some of them 7p15, leading to the formation of a JAZF1/PHF1 fusion gene. display considerable molecular diversity both in terms of intragenic variant fusions and the involvement of alternative translocation The third tumor showed a t(6p;10q;10p) as the sole karyotypic abnormality, leading to the fusion of PHF1 with another partners in pathogenetically equivalent variant translocations partner, the enhancer of polycomb (EPC1) gene from 10p11; (10, 11). Accordingly, the failure to detect the JAZF1/JJAZ1 fusion EPC1 has hitherto not been associated with neoplasia. The transcript in ESS not showing the 7;17 translocation (7, 12) suggests PHF1 gene encodes a protein with two zinc finger motifs the existence of pathogenetic, cytogenetic, and molecular variants whose involvement in tumorigenesis and/or tumor progres- of the ESS-specific t(7;17) and JAZF1/JJAZF1. Indicative of the sion has not been reported before, but its rearrangement same possibility is also the fact that chromosomal band 7p15 was found rearranged in 5 of the 32 ESS with karyotypic changes but clearly defines a new pathogenetic subgroup of ESS. (Cancer without the aforementioned 7;17 translocation (8). The second Res 2006; 66(1): 107-12) most common cytogenetic abnormality registered in ESS involves chromosomal band 6p21, rearranged in eight tumors; this too Introduction hints at pathogenetic, cytogenetic, and molecular genetic ESS- specific features that have not yet been sufficiently examined and Malignant tumors mimicking the differentiation of endometrial understood. stromal cells, endometrial stromal sarcoma (ESS), are rare and We therefore subjected to detailed analysis three ESS showing account for <10% of uterine sarcomas. ESS were traditionally rearrangement of chromosomal band 6p21. In all tumors, specific divided into low-grade and high-grade tumors according to the involvement of the PHD finger protein 1 (PHF1) gene in 6p21 could classification of Norris and Taylor (1), but because the so-called be shown. PHF1 was found recombined with two different partners, high-grade ESS usually lack specific differentiation and often with the JAZF1 gene in the two tumors showing a 6p;7p rearrange- present little or no histologic resemblance to endometrial stroma, ment and with the enhancer of polycomb (EPC1) gene from 10p11 they are now mostly referred to as undifferentiated endometrial in the third tumor, which had a 6;10;10 translocation as the sole sarcomas (2). ESS therefore consist of only low-grade ESS and karyotypic abnormality. variants thereof, regardless of the mitotic index, as long as the lesional cells resemble nonneoplastic proliferative endometrial stroma (3–5). Materials and Methods Tumors. Samples from three surgically removed ESS were subjected to histologic and genetic analyses. The patient of case 1 was a 33-year-old woman who underwent hysterectomy after a biopsy of a uterine tumor had Requests for reprints: Francesca Micci, Department of Cancer Genetics, The shown an ESS. The tumor cells were small and spindle shaped with a Norwegian Radium Hospital, 0310 Oslo, Norway. Phone: 47-2-293-4436; Fax: 47-2-293- mitotic count of 0.1 per high-power field. Only small areas with fibrous or 5477; E-mail: [email protected]. I2006 American Association for Cancer Research. myxoid tissue were seen. The tumor infiltrated extensively through the doi:10.1158/0008-5472.CAN-05-2485 uterine wall and into the parametrium and vessels (Fig. 1). The patient of www.aacrjournals.org 107 Cancer Res 2006; 66: (1). January 1, 2006 Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2006 American Association for Cancer Research. Cancer Research Fluorescence in situ hybridization analyses. Different fluorescence in situ hybridization (FISH) analyses were done depending on which types of chromosome aberrations were discovered by G-banding. Multiplex FISH (15) was done on cases 1 and 2; in case 1 on a previously G-banded slide according to the protocol described by Teixeira et al. (16), whereas in case 2, fresh chromosome preparations were available for multiplex FISH. In case 3, locus-specific probes derived from bacterial artificial chromosomes (BAC) and P1 artificial chromosome clones were prepared and hybridized to detect the exact breakpoint of the 10;10 translocation originally thought to be the only chromosomal abnormality present in this tumor (see below). The BAC clones were retrieved from the RPCI-11 Human BAC library and CalTech human BAC library D; the P1 artificial chromosome clones were from the RPCI-4 human P1 artificial chromosome library (P. de Jong libraries, http://bacpac.chori.org/home.htm). They were selected according to physical and genetic mapping data on chromosomes 6, 7, and 10 (see below) reported in the Human Genome Browser at the University of California, Santa Cruz website (May 2005, http://genome.ucsc. edu/). The clones initially used were RP11-414H17 and RP11-74N14 mapping to 10p15 and RP11-34E5 and RP11-7D5 mapping to 10q24. The identification of a three-way translocation involving also chromosome 6 and the redefinition of the breakpoints on 10p and 10q (see below) led us to perform additional analyses using BAC clones RP11-615A19, RP11-513I15, and RP11-754H10, mapping to 6p21 (from centromere to telomere); RP11- 322I2 and RP11-241I20 mapping to 10p11; and 433J16 mapping to 10q21 (resources for Molecular Cytogenetics, Bari, Italy; http://www.biologia. Figure 1. Histologic sections of the three ESS. A, extensive infiltration in the uniba.it/rmc/). In case 2, clones RP11-81H15 and RP4-781A18, mapping to myometrium in case 1. B, vessel infiltration in case 3. C, tumor with a finger-like projection into the surrounding myometrium in case 2. D, extensive 7p15 and covering the JAZF1 locus, and CTD-2307H19, mapping to 6p21 hyalinization with areas of endometrial stromal cells around spiral arteriole-like and covering the PHF1 locus, were used to detect JAZF1/PHF1 fusion at the vessels in case 2. molecular cytogenetic level. The clones were grown in selective media, and DNA was extracted according to standard methods (17). DNA probes were case 2 was a 72-year-old woman with a 2.2-cm large tumor in the anterior directly labeled with a combination of FITC-12-dCTP and FITC-12-dUTP, wall of the uterine corpus. Histologic examination of the operation Texas Red-6-dCTP and Texas Red-6-dUTP (New England Nuclear, Boston, specimen showed a tumor with large areas of hyalinization as well as MA), or indirectly with biotin-dUTP (Molecular Probes, Invitrogen, Carlsbad more cellular areas with spindle cells without atypia. The mitotic count was CA) by nick translation and detected with streptavidin-diethylaminocou- 0.1 per high-power field. The tumor was well demarcated macroscopically marin (Invitrogen). The subsequent hybridization conditions as well as the but microscopically showed one or two finger-like tumor projections detection procedure were according to standard protocols (18). exceeding 3 mm into the surrounding myometrium (Fig. 1). No infiltration Molecular genetic investigations. Tumor tissue adjacent to that used of vessels was seen. The patient of case 3 was a 34-year-old woman who for cytogenetic analysis and histologic examination had been frozen and underwent hysterectomy because a biopsy had shown the presence of stored at À80jC. Total RNA was extracted from the three tumors using ESS. Macroscopic examination showed a polyp-like tumor with a diameter of Trizol reagent according to the manufacturer’s instructions (Invitrogen). 2.5 cm in the uterus.